The TMSD Threshold System (TTS) is a modular threshold system designed to provide a clear, binary output signal in response to subtle changes in input RNA concentration. It relies on Toehold-Mediated Strand Displacement (TMSD), where RNA strands A, B, and C interact to create a tuneable threshold. Strand A serves as the input, binding to strand B via a toehold and displacing strand C once the concentration of A exceeds the threshold of free strand B. Displaced strand C then triggers strand D, which contains an inducible spinach aptamer that refolds into a fluorescent form, generating a measurable output. This system ensures precise detection by only producing a signal when RNA levels surpass a set threshold, making it ideal for cell free diagnostic applications. We have added the whole TMSD Threshold System to the iGEM Parts Registry, as a part collection of composite parts (BBa_K5106015, BBa_K5106016, BBa_K5106017, BBa_K5106018).
Overview TMSD and Threshold
In patients with autoimmune illnesses, there are signs of inflammation that can be detected in the blood, such as the up- or downregulation of miRNA (Figure 1a). These dysregulated miRNA patterns can be used to diagnose patients. However, some of these miRNA dysregulations are subtle, as little as a 5% marginal increase in concentration have been cited as significant in correlation with diseases.1 This is a problem because toehold switches increase their output linearly (Figure 1b), meaning a small increase in input will result in a small change in output. This causes an intermediate output rather than a binary one (Figure 1b). A binary output (being ON or OFF) is preferred because this gives a clear signal for MS diagnosis. To distinguish these minor changes in miRNA concentration in patients, a threshold detection system is required. A threshold would mean that only if the miRNA is above a certain concentration, an output signal can be produced (Figure 1b). Since different miRNAs can have different concentrations in the blood, the level of the threshold must be tuneable. This means that for any miRNA, regardless of sequence or concentration, a concentration threshold can be constructed.
One method to construct such a tuneable threshold, is by utilising the properties of toehold mediated strand displacement (TMSD). TMSD is the process of replacing an annealed RNA strand, with one that has a higher binding affinity. This preferential affinity of RNA strands with better annealing, can create a sharp transition in output signal (Figure 1b).
In TMSD there are at least three strands of RNA present: A, B and C (Figure 2a). In the initial state, strand B and C are annealed, however strand A has a higher affinity for B. Because there is a free region of RNA on strand B, strand A can partially bind. This is known as the toehold, and is essential to stabilise the kinetic bottleneck in the reaction. When the toehold is at least six bases long, the forward reaction kinetics in the exchange reaction improve, with equilibrium constant K increasing up to the power six.2 Once partially bound, strand A can compete with strand C, ending in strand C being displaced from strand B. To create a TMSD threshold you need three RNA strands where all binding affinities are controlled so that the binding energy of RNA pairs is ranked: AB>BC>CD, with D being an external output receptor for C. This is because A must anneal stronger to B, otherwise a very large ratio of A:C is required to displace C.
This alone does not create a threshold, for this we need a surplus of strand B. By creating a pool of free strand B, low levels of strand A will bind to these, not releasing any of strand C. By controlling the concentration of free B, a threshold can be created. In the situation where there is less A present than B, indicated as the OFF state (Figure 3a), A binds to B, but due to the excess of B, no C is released. When A is present in higher concentrations (Figure 3b), the unbound B is sequestered and C is displaced from B. This means output signal C is only released when A is present in concentrations above the set threshold.
The slope of this threshold is dependent on the ratio between B and C. If the ratio of B to C is 1:1 when any A is present, C will be released, causing a linear relation between A and C (Figure 4a). However, when the ratio of B is increased, a larger fraction of A is required to bind the free B than to release the C (Figure 4b). The smaller the fraction of A that releases C becomes, the more the output signal starts to resemble a full binary signal. Computer modelling has shown that a B:C ratio larger than 3 can create a near binary output response (Figure 4c).
This system allows for an easy and modular system for thresholding in cell-free systems. The RNA sequences can be easily designed based on a desired input and/or output sequence, and the threshold can be altered by simply changing the concentration RNA strand B. The output signal C can modified for any downstream detector (D), which can then detect the output signal of the threshold system C. This downstream detector D can be a toehold or an inducible fluorescent aptamer, thereby giving a visible signal in a Cell-free test.
Spinach
In the chosen system, the output C was visualised by using an inducible spinach aptamer produced by Wang et al. (2023).3 Spinach is an RNA structure that can stabilise the side group of a fluorophore molecule (DFHBI), which allows it to fluoresce. The inducible spinach structure (Figure 5) contains a misfolded spinach aptamer, that upon binding a trigger (in this case C), can refold into a fluorescing structure that can be quantified in a fluorescence microplate reader. By measuring the fluorescence in a plate reader, the output of C can be detected. Since the structures of D and C are set due to the refolding requirements of D, A and B had to be adapted to comply with these requirements.
NUPACK
NUPACK is a software that can calculate the hybridising energies of all possible RNA structures and select the thermodynamic optimal, and therefore most statistically likely structure.4 NUPACK was used to model affinities of desired pairs and prevent undesired binding between unrelated RNA sequences. Based on a template form of D (an inducible spinach aptamer in this context) and the associated trigger C from a paper by Wang et al. (2023),3 the template parts A and B were designed. Based on the sequence C, an antisense structure B was designed with a greater affinity to from BC than CD and can generate a sharp threshold which binding affinities that follow AB>BC>CD. C was elongated to improve the hybridisation between AB and consequent TMSD. Strand B was designed complementary to C, with an additional toehold and elongated region to hybridise with A.
Once the parts A-D were designed (Figure 6), they were analysed in NUPACK to confirm the correct binding preference (Figure 7). Results of the equilibration showed that in conditions where the concentration of A exceeds that of B, C and D hybridise (Figure 7a). In conditions where the concentration of A is lower or equal to B, C and D cannot hybridise (Figure 7b). This means that the threshold system functions as predicted.
The system as proposed above is a versatile thresholding system, with applications to many cell-free systems. As has been shown in the example case above, the system can be designed for any output system D, such as a toehold. When this threshold system is combined with toehold circuits and logic gates, by replacing D in the system, it can provide an extra layer of information for each of the inputs of the circuit. Which in the case of miRNA for the miRADAR test, is a critical part. As mentioned before miRNA is constantly present, and the change in concentration is too subtle for many detection methods. miRADAR using this threshold, proposes a revolutionary fully integrated quantifiable test for miRNA.
Exerimental design and results
For establishing the threshold of miRNAs in our test, we used a Toehold-Mediated Strand Displacement (TMSD) threshold system, further referred to as TTS. The TTS system, consisting of the four different parts, acts as one part collection, which consists of basic parts BBa_K5106011-BBa_K5106014, integrated into composite parts BBa_K5106015 - BBa_K5106018. After we designed and confirmed the sequences of the TTS in NUPACK, we converted them into DNA and combined them with a T7 promotor. This allowed us to order the sequences as DNA oligos and transcribe them using in vitro transcription (IVT) back into RNA. The T7 promotor allows RNA polymerase to bind and amplify the RNA sequence at 37°C. This was done in a thermocycler to ensure optimal temperature and minimal evaporation. RNA from IVT was directly loaded in a 96-black clear bottom plate for fluorescence detection. No purification was performed as this caused irredeemable misfolding of the fluorescent aptamer. Spinach-DFHBI-1t fluorescence was measured at 470/505 nm at 25°C, 30 minutes after adding the RNA fragments to allow the mixture to reach equilibrium.
Alternatively, RNA was transcribed in a plate reader at 37°C to directly measure the fluorescence and thereby the concentration of RNA in the system. DNA with IVT buffer and enzymes was directly loaded in a 96-well black clear bottom plate for fluorescence detection. To obtain a fluorescent signal, C and D had to be co-transcribed to result in the correct folding of the fluorescent aptamer. Spinach-DFHBI-1t fluorescence was measured at 470/505 nm at 37°C in 30-minute intervals, to reduce photobleaching.
We observed that fluorescence was produced in the presence of TTS part D alone (Figure 8), this background signal was to be expected. Since it was less than 20% of the signal of CD, this was deemed acceptable. Notably more fluorescence was observed when part C and D were co-transcribed in the same system, indicating that co-transcription is necessary to ensure correct folding. When fragment C is produced separately, no fluorescent output signal was observed as expected. This was also the case for the negative control. The positive control sample consisting of the spinach-2 fluorescent aptamer also showed no fluorescence. This was hypothesised to be caused by misfolding of the RNA during IVT. This was shown by heat denaturation of the spinach-2 aptamer, after which the RNA was able to refold, and fluorescence was restored (not shown).
After the first experiment in which we showed that TTS part C and D can produce fluorescence, RNA strand A was added to the reaction mix. This was to produce a threshold, opposite of that originally designed, where the subsequent addition of B would inactivate the fluorescence. Since A should only have affinity for strand B, no change was expected. However, an almost 3-fold higher fluorescent signal was observed after the addition of A (Figure 9). Since A itself cannot bind and stabilise DFHBI-1T, it was speculated that it might have affinity for D.
This was confirmed by a NUPACK analysis (Figure 10), which indicated that A could bind with D. This was missed since in previous analyses C and D were expected, and therefore modelled, to be at equal concentration. If the concentration of D is greater than that of C, A can bind to D. Since NUPACK showed these criteria are essential for this reaction to occur, we must assume this is what caused the increase in fluorescence due to the addition of A (Figure 10). This is in line with protocols about IVT, which states that longer RNA sequences transcribe more efficiently than short RNA sequences. Therefore we hypothesise that; 1: Due to the longer DNA sequence, fragment D has been transcribed more.; 2: Due to the anti-sense compatibility A can bind to D (when C is present in smaller concentrations than D), and induce fluorescence.
To test the full functionality of the TTS system, we investigated how well part B could inhibit the fluorescent signal caused by part C, D and A. This is a simplified (and reverse) version of the TTS system, as it was unsure whether C could again bind to D and produce fluorescence after release by B. This system was predicted give a similar threshold reaction, however the threshold would be based on the concentration A, and the signal would be inhibited once the concentration of B exceeded that of A.
Since the binding affinity between part B and C (and especially A and B) is higher than between part C and D, addition of part B should lead to a decrease in the observed fluorescence. The fluorescence of CD was measured at 470/505 nm with varying concentrations strand B (Figure 11). It was observed that fluorescence indeed went down over time with the addition of part B. In addition, fluorescence went down to a plateau that was also determined by the concentration of inhibitor B, whereby adding more B resulted in lower fluorescence. This indicated that part B can indeed inhibit fluorescence. part C as expected, and can thus be used for creating the TTS system together with part C and D.
However, since the fluorescence of all wells dropped immediately without any threshold function being observed, therefore we hypothesise that C/A has not saturated all of D, therefore when any B is present, CD/AD pair is split up and fluorescence decreases.
Altogether, we showed that fluorescence caused by the secondary structure formed by part C and D together works. In addition, inhibition of part B is also successful. The next step would be proving that the inhibition of B, only occurs once the threshold of A is exceeded. Secondly it must be shown that A can release part B again as required for the complete TTS system.
Redesign
We learned that A can bind to D, and that therefore, the ratio of strands C and D is of great importance for a working threshold. To obtain this predicted optimal ratio of C and D in the lab, the co-transcription of C and D could be optimised. This could for instance be done by altering the DNA template concentration in the IVT reaction. Alternatively, since CD fluorescence can be increased by addition of A, it stands to reason that same would hold for addition of C. Thereby C could be added until the fluorescence no longer increases (when the concentration of C exceeds that of D). This way D, would be saturated with C and no A could bind. By doing this, the original threshold experiment as mentioned below (Figure 11) above could be tested again. However, due to a lack of time, we were not able to perform this experiment again.